Insulin formulations and methods of using same in preterm infants

ABSTRACT

An insulin formulation and a method of preparing and using same. The insulin preparation includes insulin, oligosaccharide and sodium chloride at a ratio of 1:A:B (w:w:w respectively) with ranges of A and B are 1000-5000 and 10-50, respectively.

RELATED APPLICATIONS

This application is a Continuation of PCT Patent Application No.PCT/IL2022/050371 having International filing date of Apr. 11, 2022,which claims the benefit of priority under 35 USC § 119(e) of U.S.Provisional Patent Application No. 63/173,442 filed on Apr. 11, 2021.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to insulin formulation formulated for usein infant feed. Embodiments of the present invention relate to methodsof preparing insulin and an infant feed including same and to methods ofusing the insulin-containing infant feed in preventing or reducingseverity of pre-term infant disorders such as late onset sepsis ornecrotizing enterocolitis/colitis/enterocolitis.

Birth is considered premature, or preterm, when it occurs before the37th week of pregnancy. The final weeks in the womb are crucial forhealthy weight gain and for the full development of various vitalorgans.

Human milk is recognized as the optimal feeding for all infantsincluding pre-term infants because of its proven health benefits toinfants.

Human milk contains a substantial number of hormones and growth factorsand studies have shown that some of these hormones (e.g. insulin,insulin-like growth factor 1, IGF-1, epidermal growth factors) have aneffect on the small intestine following oral administration.

Studies have also shown that that addition of insulin to preterm infantformulas results in better growth and accelerated intestinal maturation[Shulman, R. J., 2002; Archives of Disease in Childhood-Fetal andNeonatal Edition, 86(2), F131-F133].

“Insulin-containing infant feed formulations have been previouslydescribed. However, when infants are born prematurely, their digestivesystems may not be fully developed and as a result, many of thesepre-term infants experience feeding difficulties and disorders thatdirectly or indirectly relate to malnourishment.”

Late-onset sepsis (LOS), defined as sepsis onset after 72 h of life, isa leading cause of mortality in the neonatal intensive care unit. Theincidence rates for LOS in preterm infants vary between 20 and 38% inthe first 120 days of life, and mortality rates range from 13 to 19%.

Necrotizing enterocolitis (NEC) is a disease that affects mostly theintestine of premature infants. The wall of the intestine is invaded bybacteria, which cause local infection and inflammation that canultimately destroy the wall of the intestine. About 7% of pre-terminfants develop necrotizing enterocolitis and among those affected,about 25% die.

While reducing the present invention to practice, the present inventorshave formulated an insulin formulation that can be solubilized in aninfant feed (formula or breast milk) and have shown for the first timethat such a feed formulation can be used to prevent, or reduce theseverity of LOS and NEC.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of preparing insulin for use in an infant feed comprising:solubilizing insulin in hydrochloric acid to obtain solubilized insulin;and diluting the solubilized insulin with water and adjusting a pH to6.9-8.0.

According to embodiments of the present invention the method furthercomprises adding cyclodextrin to solution (b) to thereby encapsulate theinsulin in cyclodextrin cavities; and adjusting pH to 6.9-8.0.

According to embodiments of the present invention the method furthercomprises compounding a result of (b) or (d) with a sodium chloridesolution and a maltodextrin solution and adjusting the pH to 6.7-7.6.

According to embodiments of the present invention the composition iscombined with an oil-in-water nanodroplets emulsion wherein thenanodroplets include the Docosahexaenoic acid (DHA) and the coenzymeQ10.

According to embodiments of the present invention the method furthercomprises filtering a result of the compounding and lyophilizing afiltrate to obtain an amorphous powder with a pH 6.0-7.6

According to another aspect of the present invention there is provided amethod of producing an infant feed with insulin comprisingreconstituting the amorphous powder described herein in the infant feed.

According to another aspect of the present invention there is providedan insulin formulation comprising insulin, oligosaccharide and sodiumchloride at a ratio of 1:A:B (w:w:w respectively), wherein ranges of Aand B are 1000-100,000 and 10-50, respectively.

According to embodiments of the present invention the insulinformulation has a pH of 6.0-7.6 when reconstituted in deionized water or0.9% sodium chloride solution at concentration of 0.03 g/ml.

According to embodiments of the present invention the insulinformulation further comprises cyclodextrin.

According to embodiments of the present invention the cyclodextrin is ata ratio of 10-1000 with respect to the insulin.

According to embodiments of the present invention 1 gram of the insulinformulation includes 2-40 IU.

According to another aspect of the present invention there is providedan infant feed comprising the insulin formulation.

According to another aspect of the present invention there is provided amethod of preventing necrotizing enterocolitis/colitis/enterocolitis ina preterm infant comprising administering to the preterm infant theinfant feed.

According to another aspect of the present invention there is provided amethod of reducing the prevalence and severity of necrotizingenterocolitis/colitis/enterocolitis in a preterm infant comprisingadministering to the preterm infant the infant feed.

According to another aspect of the present invention there is provided amethod of reducing the prevalence of late-onset sepsis in a preterminfant comprising administering to the preterm infant the infant feed.

According to another aspect of the present invention there is provided amethod of reducing inflammation in the gastrointestinal in a preterminfant comprising administering to the preterm infant the infant feed.

According to another aspect of the present invention there is provided amethod of reducing the prevalence and severity of adverse events,life-threatening events and fatal complications in a preterm infantcomprising administering to the preterm infant the infant feed.

According to another aspect of the present invention there is provided amethod of reducing permeability of the GI tract in a preterm infantcomprising administering to the preterm infant the infant feed andmeasuring a lactose/lactulose ratio.

According to another aspect of the present invention there is provided amethod of increasing the rapid maturation of the gut microbiota in apreterm infant comprising administering to the preterm infant the infantfeed and measuring a reduction in Proteobacteria and an increase inFirmicutes and Clostridiaceae.

According to embodiments of the present invention the infant feedincludes 0.01-4 IU/ml.

According to another aspect of the present invention there is provided amethod of preventing late onset sepsis in a preterm infant comprisingadministering to the preterm infant the infant feed.

According to another aspect of the present invention there is provided amethod of reducing a discharge time from primary care of a preterminfant comprising administering to the preterm infant the infant feeddisclosed herein.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a typical chromatogram of formulations I-IV of the presentinvention.

FIG. 2 is a typical chromatogram of formulations V-VI of the presentinvention.

FIG. 3 is a typical chromatogram of DHA and Coenzyme Q10 in formulationsIV-V of the present invention.

FIG. 4 illustrates the Probability of parenteral nutrition (PN) wean-offper group (Kaplan-Meier survival curve).

FIG. 5 illustrates the average PN intake per day per group, presenting aclear benefit of the treatment group, especially for the Enteral insulinformulation at 0.3 IU/kg/day compared to placebo (shown as the rate ofPN reduction out of total intake).

FIG. 6 illustrates the percentage of enteral nutrition out of totalnutrition per group.

FIG. 7 illustrates the resents the trajectories per group of FIG. 6 .

FIG. 8 is a scanning electron micrograph showing the amorphous structureof formulations I-IV of the present invention.

FIGS. 9-10 are graphs showing thermogravimetric analysis (TGA) anddifferential scanning calorimetry (DSC) of formulations I-IV of thepresent invention.

FIG. 11 is a solid-state NMR spectrum of formulation I-IV of the presentinvention.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention is of an insulin formulation which can be used inan infant feed. Specifically, the present invention can be used toprevent or reduce severity of pre-term infant disorders such as lateonset sepsis or necrotizing enterocolitis/colitis/enterocolitis.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

Insulin-containing infant feeds are known in the art and have beensuggested for treatment of malabsorption in preterm infants.

While conducting experiments with various insulin formulations intendedfor use in infant feeds such as maternal milk or formulas, the presentinventors have observed that when solubilized in infant feed, insulinexhibits the tendency to polymerize and agglomerate thus reducing theavailability of insulin in the resultant feed formula. While vigorousmixing of the infant feed can partially resolve this problem, thepresent inventors postulated that this is a less than optimal solutionand set out to improve insulin solubility in infant feed.

Thus, according to one aspect of the present invention there is providedand insulin formulation that is readily dissolvable in an infant feed(e.g., maternal milk or formula) and exhibits stability (structural andfunctional) in the feed without agglomeration for extended periods oftime.

The present preparation includes insulin (e.g., human recombinant), acarbohydrate (e.g., oligosaccharide such as maltodextrin) and sodiumchloride at a ratio of 1:A:B (w:w:w respectively), wherein ranges of Aand B are 1000-100,000 and 10-50, respectively. One gram of the insulinformulation can include 2-40 IU. The insulin formulation can furtherinclude cyclodextrin at a ratio of 10-1000 with respect to the insulin.Carbohydrates such as, for example, monosaccharides (e.g. glucose),disaccharides (e.g. lactose, maltose), oligosaccharides (dextrins (e.g.Maltodextrin), cyclodextrins (e.g. Hydroxypropyl-beta-cyclodextrin(HPbCD) and polysaccharides are used as stabilizers, fillers andcryoprotectants. Buffering agents (e.g. sodium or potassium phosphatesalts) can also be added to the composition.

The formulation can be combined with an oil-in-water nanoemulsion havingnanodroplets that include the Docosahexaenoic acid (DHA) and coenzymeQ10.

The formulation can be in powder form and when reconstituted indeionized water or 0.9% sodium chloride solution at concentration of0.03 g/ml can exhibit a pH of 6.0-7.6.

The present inventors have discovered that pretreatment of insulin at pH6.9-8.0, compounding with a carbohydrate at pH 6.7-7.6 andlyophilization, results in an amorphous insulin powder (FIG. 8 ) that isfreely soluble in human breast milk and thus overcomes theaforementioned limitations of prior art insulin formulations.

The present insulin formulation is an amorphous powder in which insulinis incorporated into an amorphous structure mainly composed of a filler(e.g. maltodextrin). The water content of the preparation can be 6.5% orlower, the osmolarity is about 110-120 mosm/kg when reconstituted indeionized water at concentration of 0.1 g/ml and the pH is 6.0-7.6 whenreconstituted in deionized water or 0.9% sodium chloride solution atconcentration of 0.03 g/ml. Typical thermogram curves, obtained bythermogravimetric analysis (TGA) and Differential scanning calorimetry(DSC), are shown in FIGS. 9-10 . Solid-state NMR spectrum of aformulation of the present, invention is provided in FIG. 11 .

As is mentioned herein, the present insulin formulation can be added asa powder (or liquid) to an infant feed such as, for example, human milk.The human milk be mother breast milk or donor breast milk fresh andhomogenized.

When in powder form, the present insulin formulation can bereconstituted in an infant feed to a final insulin concentration of0.1-8 IU/ml.

The present formulation can be packaged as a powder and stored forextended time periods (days/months).

The package can be, for example, a glass vial or an aluminum sachetfilled with a dry form of the present formulation. The pack can include0.1-1.0 grams of a powder form of the formulation with each gramincluding 0.2-40 IU insulin. The headspace of the packaging unit can beair or inert gas (e.g. nitrogen).

The package can provide a moisture and/or oxygen barrier with a moisturevapor transmission rate (MVTR) of less than about 0.1 g/m²/24 h and anoxygen transmission rate (OTR) of less than about 0.1 cm³/m²/24 h.

When packaged as disclosed herein, the present formulation is highlystable and can be stored for prolonged periods with insulin activitydecreasing by no more than about 1-10%, when stored at between about 2°C. to about 8° C. for up to 24 months. Additionally, the water contentof the packaged formulation is typically no more than about 4.5-6.5% byweight when stored at between about 2° C. to about 8° C. for 24 months.

The insulin formulation can be reconstituted in deionized water or 0.9%sodium chloride solution and the resulting solution can be added to aninfant feed (Mother's Own Milk, Donor Breast Milk, Formula etc.) toprovide the concentration of insulin described above for the powderedformulation.

As is described herein and demonstrated in the Examples section, thepresent insulin formulation and feed containing same can be used toaccelerate wean-off of pre-term infants from parenteral nutrition and tosubstantially reduce adverse events overall, as well as specificconditions of interest including necrotizing colitis (NEC) and lateonset sepsis (LOS).

Thus, according to another aspect of the present invention there isprovided a method of preventing or reducing the risk and severity of,LOS in a subject in need such as a preterm infant. The method iseffected by administering a therapeutically effective amount of theinfant feed mixed with the drug of the present invention to the subjectin need. Such administration can be oral or through a feeding tube.

Thus, according to yet another aspect of the present invention there isprovided a method of preventing or reducing the risk and severity of,NEC in a subject in need such as a preterm infant. The method iseffected by administering a therapeutically effective amount of theinfant feed mixed with the drug of the present invention to the subjectin need. Such administration can be oral or through a feeding tube.

The term “therapeutically effective amount” denotes a dose of an activeingredient or a feed comprising the active ingredient that will providethe therapeutic effect for which the active ingredient is indicated.

As used herein the phrase “subject in need thereof” refers to a humaninfant. The human infant can be at any age (e.g., a term or preterminfant) or of any gender. The subject in need can be a preterm infantborn at a gestational age of 24 to 37 weeks or a low-birth-weight fullterm infant or an Intrauterine growth restriction (IUGR) infant or Smallfor Gestational Age infant.

A typical dose regimen established by the present inventors can beadministered via continuous enteral administration or bolus intake using0.1-1 gm of powder mixed into 1-3 ml of an infant feed divided into 1-12oral administrations a day for example.

As is described in the Examples section that follows, the presentregimen resulted in less NEC cases in the 0.3 IU/kg/day dose groupcompared to placebo; Necrotizing enterocolitis (Bell stage 2 or 3)occurred in 7 infants (6%) in the low-dose group, 4 infants (5%) in thehigh-dose group, and 10 infants (10%) in the placebo group. Whenobserving the most fragile population (26-28 weeks gestational agestrata) the difference is even more pronounced—4.3% of subjects intreatment group had NEC vs. 19.6% in the placebo group. There was ameaningful difference between groups in the percentage of infantsexperiencing infections (including sepsis and other infections). Whenobserving the entire study population, the general incidence was 30% inthe 0.06 IU/kg/day dose group, 34.7% in the 0.3 IU/kg/day dose group and42.9% in the Placebo group.

A simultaneous assessment of intestinal permeability and lactaseactivity by sugar absorption test (SAT) performed in another study usingthe present regimen showed a trend towards lower intestinal permeability(indicating rapid maturation of the GI) and higher lactase activity inthe treatment groups.

The efficacy of the present study is also demonstrated by the time todischarge (to home or secondary site) as is demonstrated by the resultsshown in Example 4. As is shown therein, a reduction of time todischarge from primary care of a preterm infant is by at least 5% ascompared to an untreated preterm infant (control). This translates todischarge that is at least 5 days earlier than that expected.

Another study was performed aimed to assess the efficacy of 2 doses ofenteral insulin administration compared to placebo with respect to earlydifferences in intestinal microbiota. This study showed significantdifferences in GI microbiota between the treatment groups and placebo.At the end of treatment, high insulin dose showed less microbialdiversity than placebo (p=0.0192). Insulin increases Firmicutes phylumand decreases Proteobacteria phylum. Enteral insulin administration hada reduction in Proteobacteria and an increase in Firmicutes andClostridiaceae. These results indicate that the present formulation andregimen facilitate rapid maturation of gut microbiota. Decrease ofProteobacteria and increase of Firmicutes obtained in this study are thechanges observed previously in low risk infants for NEC.

As used herein the term “about” refers to ±10%.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non-limiting fashion.

Example 1 Insulin and Infant Feed Formulations

Insulin formulations were prepared and tested (in an infant feed) foreffectiveness in treating preterm infants.

Formulation I

Insulin as active ingredient, sodium chloride as solubilizing agent andoligosaccharide as filler and stabilizer (e.g. Maltodextrin). Thefinished product is a lyophilized powder packed in glass vials. Strengthof the reconstituted solution is regulated by the diluent volume.

TABLE 1 Content per Ingredient unit dose (vial) Recombinant Human0.04-0.05 mg Insulin (rh-Insulin) (1-1.5 IU) Maltodextrin 100-500 mgSodium Chloride 1.0-2.0 mgMaterials and Methods

A solution of rh-Insulin, 24.5 IU/ml, was prepared as follows: 13 mgInsulin were solubilized in 1 ml 0.1N hydrochloric acid, diluted up to15 ml with water and the pH was adjusted to 7.6 with 0.1N sodiumhydroxide.

Formulation Compounding:

Forty grams of maltodextrin were dissolved in 100 ml water and pH of theobtained solution was adjusted to 7.2. 2 ml of 6% sodium chloridesolution and 4 ml of Insulin solution were added to the formulationmixture, the pH was adjusted to 7.0.

The resulting bulk was filtered through 0.22 um PES membrane, filledinto the glass vials (filling volume 1.7 ml) and lyophilized.

Each vial with lyophilized powder contained 1.25 IU rh-Insulin, 510 mgmaltodextrin and 1.5 mg sodium chloride.

Formulation II

Insulin as active ingredient, cyclodextrin as encapsulating agent (e.g.Hydroxypropyl Beta-Cyclodextrin), sodium chloride as solubilizing agentand oligosaccharide as filler and stabilizer (e.g. Maltodextrin). Thefinished product is a lyophilized powder packed in glass vials. Strengthof the reconstituted solution is regulated by the diluent volume.

TABLE 2 Content per Ingredient unit dose (vial) Recombinant HumanInsulin (rh- 0.04-0.05 mg Insulin) (1-1.5 IU) HydroxypropylBeta-Cyclodextrin 2.0 mg Maltodextrin 100 mg Sodium Chloride 1.0 mgMaterials and Methods

A solution of rh-Insulin, 62 IU/ml, was prepared as follows: 85.6 mgInsulin were solubilized in 5 ml 0.1N hydrochloric acid, diluted with 30ml water and the pH was adjusted to 7.2 with 0.1N sodium hydroxide.

Formulation Compounding:

Two hundred grams of maltodextrin were dissolved in 600 g water and pHof the obtained solution was adjusted to 7.3. 2 g of sodium chloride,pre-dissolved in 60 ml water, and Insulin solution were added to theformulation mixture, the pH was adjusted to 7.0 and additional portionof water was added to complete the bulk volume to 1 L.

The resulting bulk was filtered through 0.22 um PES membrane, filledinto the glass vials (filling volume 0.5 ml) and lyophilized.

Each vial with lyophilized powder contained 1.2 IU rh-Insulin, 100 mgmaltodextrin and 1.0 mg sodium chloride.

Formulation III

A solution of rh-Insulin, 243 IU/ml, was prepared as follows: 84.4 mgInsulin were solubilized in 1 ml 0.1N hydrochloric acid, diluted up to10 ml with water and the pH was adjusted to 8.1 with 0.1N sodiumhydroxide.

Formulation Compounding:

Thirty grams of maltodextrin were dissolved in 60 ml water and pH of theobtained solution was adjusted to 7.1. 2.7 ml of 7.5% sodium chloridesolution and 1 ml of Insulin solution were added to the formulationmixture, the pH was adjusted to 7.3 and additional portion of water wasadded to complete the bulk weight to 100 g.

The resulting bulk was filtered through 0.22 um PES membrane, filledinto the glass vials (filling volume 0.5 ml) and lyophilized.

Each vial with lyophilized powder contained 1.4 IU rh-Insulin, 172 mgmaltodextrin and 1.2 mg sodium chloride.

Formulation IV

This example describes the formulation produced at commercial scale 9 kgof the liquid bulk (0.9 kg of the lyophilized powder). Insulin as activeingredient, sodium chloride as solubilizing agent and oligosaccharide asfiller and stabilizer (e.g. Maltodextrin). The finished product is alyophilized powder packed in glass vials. Strength of the reconstitutedsolution is regulated by the diluent volume.

TABLE 3 Content per Ingredient unit dose (vial) Recombinant Human 0.035mg Insulin (rh-Insulin) (1 IU) Maltodextrin 99 mg Sodium Chloride 1 mgTotal weight 100 mgMaterials and Methods

A solution of rh-Insulin, 33 IU/ml, was prepared as follows: 0.39 gInsulin were solubilized in 20 g 0.1N hydrochloric acid, diluted with300 g water for injection and then neutralized with 20 g of 0.1N sodiumhydroxide. The pH was adjusted to 7.2 with 0.1N sodium hydroxide.

Formulation Compounding:

856 grams of maltodextrin and 8.6 gram of sodium chloride were dissolvedin 6854-gram water and pH of the obtained solution was adjusted to 7.2.The insulin solution was added to the compounding mixture, the weight ofthe resulting formulation was adjusted up to 9.0 kg and the pH wasadjusted to 7.0.

The resulting bulk was filtered through 0.22 um PES membrane, filledinto the glass vials (filling volume 1 g) and lyophilized.

Each vial with lyophilized powder contained HU rh-Insulin, 99 mgmaltodextrin and 1 mg sodium chloride.

Analytical characteristics of the obtained dry powder are: content ofInsulin (insulin+A21 desamido insulin) is 1.13 IU/vial, content of A21desamido insulin is 0.5% (by area percent), content of other totalrelated substances is 0.1% (by area percent), relative standarddeviation of insulin content per vial (uniformity) is 0.9%, watercontent is 0.8%, pH is 6.7, reconstitution time of the powder in wateris below 5 minutes.Formulation V

This formulation was produced by forming a DHA and Coenzyme Q10 emulsionusing a solvent displacement method, adding rh-Insulin andcryoprotectant (e.g. dextrins, cyclodextrins), filtrating andlyophilizing.

TABLE 4 Content per unit Ingredient dose (vial) Recombinant Human 0.04mg Insulin (rh-Insulin) (1.2 IU) Cis-4,7,10,13,16,19- 2.5 mgDocosahexaenoic acid (DHA) Coenzyme Q10 (CoQ10) 1.1 mg Tyloxapol 0.5 mgLipoid E 80 0.5 mg Polyvinyl alcohol (PVA) 0.5 mg Maltodextrin 33 mgSodium hydroxide* — Hydrochloric acid* — *Sodium hydroxide orhydrochloric acid are used for adjusting the pH value and not includedin the sum.Materials and Methods

A solution of rh-Insulin, 40 IU/ml, was prepared as follows: 27.5 mgInsulin were solubilized in 2 ml 0.1N hydrochloric acid, diluted with 18ml water and the pH was adjusted to 7.2 with 0.1N sodium hydroxide.

An oil in water nanoemulsion (average droplet size 120 nm) was preparedas follows: 690 mg of Coenzyme Q10, 1533 mg of DHA ethyl ester, 300 mgTyloxapol and 300 mg Lipoid E80 were dissolved in 56 ml Ethanol. Themixture was added dropwise through 21 G needle to 300 ml of 0.1% PVAaqueous solution continuously mixed at 350 RPM at room temperature.Resulting emulsion was mixed for additional 10 minutes, diluted with0.1% PVA solution and the organic solvents were removed via tangentialflow filtration using Hydrosart, MWCO 30,000 membrane.

Ten grams of maltodextrin were dissolved in 160 ml of the obtainedemulsion and then mixed with Insulin solution. The volume of the finalbulk was completed to 300 ml and the pH was adjusted to 6.9. Theemulsion was filtered through 0.22 um PES membrane, filled into glassvials (filling volume 1 ml/vial) and lyophilized. Each vial contains 1.2IU rh-Insulin, 2.5 mg DHA and 1.1 mg Coenzyme Q10. The average size ofthe droplet particles in the reconstituted powder is 295 nm.

Formulation VI

Formulation steps are identical to those of formulation V.

TABLE 5 Content per unit Ingredient dose (vial) Recombinant Human 0.04mg Insulin (rh-Insulin) (1.2 IU) Cis-4,7,10,13,16,19- 2.3 mgDocosahexaenoic acid (DHA) Coenzyme Q10 (CoQ10) 1.1 mg Tyloxapol 0.5 mgLipoid E 80 0.5 mg Polyvinyl alcohol (PVA) 0.5 mg Hydroxypropyl BetaCyclodextrin 30 mg Sodium hydroxide* — Hydrochloric acid* — *Sodiumhydroxide or hydrochloric acid are used for adjusting the pH value andnot included in the sum.Materials and Methods

A solution of rh-Insulin, 40 IU/ml, was prepared as follows: 27.2 mgInsulin were solubilized in 2 ml 0.1N hydrochloric acid, diluted with 18ml water and the pH was adjusted to 7.2 with 0.1N sodium hydroxide.

An oil in water nanoemulsion (average droplet size 120 nm) was preparedas described for formulation IV.

Ten grams of Hydroxypropyl Beta Cyclodextrin were dissolved in 160 ml ofthe obtained emulsion and then mixed with Insulin solution. The volumeof the final bulk was completed to 327 ml and the pH was adjusted to6.9. The emulsion was filtered through 0.22 um PES membrane, filled intoglass vials (filling volume 1 ml/vial) and lyophilized. Each vialcontains 1.2 IU rh-Insulin, 2.5 mg DHA and 1.1 mg Coenzyme Q10. Theaverage size of the droplet particles in the reconstituted powder is 143nm.

Example 2 Testing of Formulations I-IV

The content of rh-Insulin was determined by reverse-phase highperformance liquid chromatography method on Hypersil Gold-C18 column(3×50 mm, 3 μm) and gradient elution with a mobile phase composed ofaqueous and organic phases (acetonitrile) acidified with 0.1%trifluoroacetic acid (TFA). Flow rate—0.3 ml/min, UV detection at 205nm. A typical chromatogram of formulations I-IV is shown in FIG. 1 .

Testing of Formulations V-VI

The content of rh-Insulin, DHA and Coenzyme Q10 was determined byreverse-phase high performance liquid chromatography method on HypersilGold-C18 column (3×50 mm, 3 μm) and gradient elution with a mobile phasecomposed of aqueous and organic phases (mixture ofacetonitrile:methanol:2-propanol 75:15:10) acidified with 0.1%trifluoroacetic acid (TFA). Flow rate—0.6 ml/min, UV detection at 205 nmfor Insulin and 214 nm for DHA and Coenzyme Q10. A typical chromatogramof formulations V-VI is shown in FIG. 2 . A typical chromatogram of DHAand Coenzyme Q10 determination in formulations V-VI is shown in FIG. 3 .

Example 3 Infant Feed Solubility

A study was designed to assess solubility of the present formulation ata pH range of human breast milk. Three dry samples, containingmaltodextrin, sodium chloride and insulin (1 IU) were prepared asfollows.

Sample 1 is a crystalline, thermally dried powder, prepared by sprayinga solution of maltodextrin, sodium chloride and insulin onto solidmaltodextrin particles and subsequent drying at 35° C. Dried powder wasmixed 1:3 with maltodextrin, the final pH was 6.3.

Sample 2 is one variant of the present formulation, presented as anamorphous powder, at pH 7.0, prepared as described in Example 1,Formulations I-III.

Sample 3 is a physical mixture of dry ingredients (maltodextrin, sodiumchloride and insulin), dry blended without any pretreatment.

A certain amount of each formulation (equivalent to 1 IU of insulin) wasreconstituted in 1.8 ml of 0.1M phosphate buffer at pH 6.3, 6.7 and 7.2and visually examined.

The results show that Sample 2 (the present formulation) dissolvesfreely in the wide pH range of breast milk (6.3-7.2), resulting in aclear solution comparable to the reference buffer. Sample 1 (thermallydried formulation) and Sample 3 (physical blend of dry ingredients, nopretreatment) were partially soluble in the tested pH range, resultingin a cloudy solution.

The dissolution profile of the test samples was evaluated using HPLC inthe following media:

0.01 N. HCl, double deionized water and 0.1 M phosphate buffer at pH 6.3and 7.4.

For each sample, the percentage of dissolution in water and phosphatebuffer was calculated relative to the amount of insulin determined in0.01 N. HCl. The dissolution results obtained (Table 6 below) indicatethat the present composition is highly soluble in the pH range of infantformula.

TABLE 6 % Dissolution of Insulin Sample 3 Sample 2- (physical blendSample 1 present of dry (thermal formulation ingredients, no Dissolutionmedium drying) (freeze drying) pre-treatment) 0.01 N HCl 100.0% 100.0% 100% Double deionized water  84.0%  93.1% 68.1% 0.1 M Phosphate buffer, 78.9%  93.3% 81.8% pH 6.3 0.1 M Phosphate buffer,  84.3%  93.6% 95.0%pH 7.4

In addition, samples 1 and 2 were reconstituted in cow's milk (pH 6.89,fat content 3%). The results were consistent with observations of thebuffer recovery test. Sample 1 is partially reconstituted in milk,resulting in visible clots that can adhere or block the passage of thereconstituted drug through the nasogastric tube. Sample 2 is completelyreconstituted in milk, resulting in a homogeneous mixture.

Example 4 Pre-Term Infant Study—Efficacy

A study was designed to assess the efficacy and safety of an enteralrh-insulin formulation in preterm infants. Following screeningprocedures, eligible infants born between 26 and up to 32 weeks ofpregnancy and weighing at least 500 g at birth were to be randomlyassigned to 1 of the 3 treatment groups in a 1:1:1 ratio.

Treatment commenced at postnatal age of at least 6 hours through andincluding 120 hours post birth. The treatment period for infants whowere fed solely on own mother's milk (OMM) commenced within the first 72hours post birth.

The treatment period was defined as the first day of dosing (Day 1) forup to 28 days or until discharge from primary hospital, if prior to Day28.

The tested formulation was administered at 0.3 IU/Kg/Day for up to 28days. Prior to administration, the powder was reconstituted mostly inhuman breast milk, donor breast milk, saline or half saline.

Results

Infants were discharged to either home or to secondary site from primaryhospital. When analyzing time to discharge, two analyses arepresented: 1) analysis of discharge from primary recruiting hospital(separate comparisons of subjects transferring to secondary hospital andsubjects discharged home); and 2) discharge to home (either from primaryor secondary hospital).

Time to Discharge from Primary Hospital

Infants were discharged from primary hospital to either home or to asecondary hospital, depending mostly on region. Each infant in the ITTanalysis had one destination for analysis, by that each arm and stratawere compared. Both treatment arms had significantly shorter time todischarge compared to placebo, demonstrating faster time to stablewell-being.

The Median number of days to discharge from hospital (to either home orsecondary hospital) was lowest in the insulin formulation comprising 0.3IU/Kg group [median=42.0 days, P=0.055], followed by the Insulinformulation comprising 0.0406 IU/Kg group [median=43.0 days, p=0.04*]compared to placebo [median=49.5 days].

Time to Discharge to a Secondary Site

Some hospitals discharge infants to secondary sites after infantsstabilize and are out of immediate life threatening condition. Infantsare discharged to secondary sites and several weeks later—to home. Inthe trial, subjects in the Insulin formulation comprising 0.3 IU/Kggroup were discharged earlier than patients in the placebo group to asecondary site—as indicated by the medians and difference from placebo(Table 7 below).

TABLE 7 Number of Days to Discharge from Primary Hospital to a SecondarySite by Gestation Age - Full Analysis Set Treatment Enteral rh- Enteralrh- insulin insulin formulation formulation Gestation 0.06 IU/kg/day 0.3IU/kg/day Placebo Age Group Statistic (N = 108) (N = 94) (N = 98) 26 -28 weeks n 14 16 14 Median 34.0 31.5 39.0 Difference 3.5 5.3 — fromplacebo 29 - 32 weeks n 8 5 12 Median 22.0 11.0 23.0 Difference −2.3 9.0— from placeboTime to Discharge to Home

Another type of analysis performed assessed the time to discharge homefor all infants, regardless of if the discharge was directly fromprimary hospital or followed a secondary site/unit. This analysis showeda similar trend for reduction in time to discharge, however showed muchlarger variability compared to the main analysis ‘discharge from primaryhospital’.

Relative to placebo, treatment with Insulin formulation comprising 0.06IU/Kg resulted in significantly fewer days to discharge to home[median=48.0 days vs median=60.0 days in placebo, P=0.025*]. Treatmentwith Insulin formulation comprising 0.3 IU/Kg resulted in a median of55.0 days to discharge to home [p=0.27 versus placebo].

When analyzing both treatment arms together, treatment groups dischargewas earlier [median=52 days, p=0.046] compared to placebo [median=60days].

Time to Wean-Off PN

A statistically significant difference in time to weaning off parenteralnutrition (PN) was observed in the group fed an insulin-supplementedfeed at a concentration of 0.3 IU/kg/day compared to placebo (6 vs. 8days, p=0.0096**).

In assessing the time to wean-off PN by gestation age group, in theolder population (29-32 weeks) time to weaning off PN was also shorterin the 0.3 IU/kg/day group (4 vs. 6 days). The results are summarized inTables 8-10 below and graphically presented in FIG. 4 which shows theProbability of PN wean-off per group (Kaplan-Meier survival curve).

TABLE 8 Analysis Summary of Number of Days to Wean-off PN- Full AnalysisSet Enteral rh- Enteral rh- insulin insulin Gestation formulationformulation Age 0.06 IU/kg/day 0.3 IU/kg/day Placebo Group Statistics (N= 108) (N = 94) (N = 98) All n (%) 107 92 97 Median 7.0 6.0 8.0

TABLE 9 Analysis Summary of Number of Days to Wean-off PN- Full AnalysisSet Gestation Treatment Stratified Van Elteren Age Group Comparison Testp-value All Enteral rh- 0.48 insulin formulation 0.06 IU/kg/day (N =107) vs. Placebo (N = 97) Enteral rh- 0.0096** insulin formulation 0.3IU/kg/day (N = 92) vs. Placebo (N = 97) Note: stratification factor:gestation age group (26-28 weeks, 29-32 weeks)

TABLE 10 Analysis Summary of Number of Days to Wean-off PN by GestationAge - Full Analysis Set Enteral rh- Enteral rh- insulin insulinformulation formulation Gestation 0.06 IU/kg/day 0.3 IU/kg/day PlaceboAge Group Statistics (N = 108) (N = 94) (N = 98) 26 - 28 weeks n 48 5951 Median 9.0 9.0 9.0 29 - 32 weeks n 59 47 46 Median 6.0 4.0 6.0

Another important endpoint for this study is the percentage of PN out ofthe total intake of the infant, as well as the gradual reduction inpercentage of PN intake over time.

FIG. 5 shows the average PN intake per day per group, presenting a clearbenefit of the treatment group, especially Enteral rh-insulinformulation 0.3 IU/kg/day compared to placebo in the rate of PNreduction out of total intake.

FIG. 6 presents the percentage of enteral nutrition out of totalnutrition per group while FIG. 7 presents the Trajectories per group.

Example 5 Pre-Term Infant Study—NEC and LOS

The study population was also observed for adverse events such asnecrotizing colitis (NEC) and late onset sepsis (LOS).

Necrotizing Colitis (NEC)

There was a meaningful difference between groups in the percentage ofinfants experiencing NEC. When observing the entire study population,general incidence was 6.3% in the 0.06 IU/kg/day dose group, 4.3% in the0.3 IU/kg/day dose group and 10.2% in the Placebo group resulting in 60%less NEC cases in the 0.3 IU/kg/day dose group compared to placebo.

When observing the younger study population (26-28 weeks GA) where NECis more prevalent, differences becomes even more apparent 12.2% in the0.06 IU/kg/day dose group, 4.3% in the 0.3 IU/kg/day dose group and19.6% in the Placebo group, resulting in nearly 80% less NEC cases in0.3 IU/kg/day dose group compared to placebo.

A statistical analysis was performed which compared both the incidenceand severity of NEC, using a non-parametric Kruskal-Wallis method, andshowed a meaningful statistical difference in the 26-28 GA group(p=0.077). The results of this study are presented in Tables 11 and 12below.

TABLE 11 NEC incidence X severity distribution per group all populationNEC CTCAE grade 1 2 3 4 5 Total % ENTERAL RH- INSULIN FORMULATION 0.06IU/kg/day (N = 110) Number of cases 0 3 1 1 2 7 % 0.0% 2.7% 0.9% 0.9%1.8% 6.3% ENTERAL RH- INSULIN FORMULATION 0.3 IU/kg/day (N = 95) Numberof cases 0 0 2 1 1 4 % 0.0% 0.0% 2.1% 1.1% 1.1% 4.3% Placebo (N = 98)Number of cases 0 1 3 2 4 10 % 0.0% 1.0% 3.1% 2.0% 4.1% 10.2%

TABLE 12 NEC incidence X severity distribution per group (26-28 weeksGA) NEC CTCAE grade 1 2 3 4 5 Total % ENTERAL RH- INSULIN FORMULATION0.06 IU/kg/day (N = 49) Number of cases 0 3 0 1 2 6 % 0.0% 6.1% 0.0%2.0% 4.0% 12.2% ENTERAL RH- INSULIN FORMULATION 0.3 IU/kg/day (N = 46)Number of cases 0 0 0 1 1 2 % 0.0% 0.0% 0.0% 2.1% 2.1% 4.3% Placebo (N =51) Number of cases 0 1 3 2 4 10 % 0.0% 1.9% 5.8% 3.9% 7.8% 19.6%

Poor outcome composite of occurrence of one of infections, NEC and deathwere calculated and compared between groups and shown in Table 13 below.

TABLE 13 Enteral rh- Enteral rh- insulin insulin formulation formulation0.06 IU/kg/day 0.3 IU/kg/day Placebo Category (N = 110) (N = 95) (N =98) Total subjects with 33 (30.0) 33 (34.7) 42 (42.9) infections n (%)Total subjects with 7 (6.4) 4 (4.2) 9 (9.2) NEC n (%) Total subjects 5(4.5) 1 (1.1) 4 (4.1) experienced death n (%) Total subjects with any 38(34.5) 34 (35.8) 49 (50.0) event n (%)

It was found that treatment groups had significantly lower percentage ofsubjects experiencing one of these adverse events of interest. Rateswere 34.5% in enteral Rh insulin formulation 0.06 IU/kg/day dose group,35.8% in the enteral Rh insulin formulation 0.3 IU/kg/day dose group and50% in the Placebo group. When performing pairwise comparison ofproportions—difference between in enteral Rh insulin formulation 0.06IU/kg/day to Placebo p=0.034, difference between enteral Rh insulinformulation 0.3 IU/kg/day to placebo p=0.065 (see Table 14 below).

TABLE 14 Summary of analysis - pairwise comparison of proportions ofsafety composite Chi-squared test Gestation of independence Age GroupComparison Test p-value All ENTERAL RH-INSULIN 0.034* FORMULATION 0.06IU/kg/day (N = 110) vs. Placebo (N = 98) ENTERAL RH-INSULIN 0.065FORMULATION 0.3 IU/kg/day (N = 95) vs. Placebo (N = 98)Late Onset Sepsis (LOS)

There was a meaningful difference between groups in the percentage ofinfants experiencing infections (including sepsis and other infections).When observing the entire study population, the general incidence was30% in the 0.06 IU/kg/day dose group, 34.7% in the 0.3 IU/kg/day dosegroup and 42.9% in the Placebo group.

Specifically, when measuring the rate of clinical or culture provensepsis, treatment groups had lower rates compared to placebo. Incidencewas 12% in the 0.06 IU/kg/day dose group, 11% in the 0.3 IU/kg/day dosegroup and 15% in the Placebo group (almost 30% reduction in late onsetsepsis incidence).

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

It is the intent of the applicant(s) that all publications, patents andpatent applications referred to in this specification are to beincorporated in their entirety by reference into the specification, asif each individual publication, patent or patent application wasspecifically and individually noted when referenced that it is to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention. To the extent that section headings are used,they should not be construed as necessarily limiting. In addition, anypriority document(s) of this application is/are hereby incorporatedherein by reference in its/their entirety.

What is claimed is:
 1. A composition-of-matter comprising an amorphousinsulin powder soluble in water, said amorphous insulin powder includinginsulin attached to dextrin particles at a gram/gram (g/g)insulin-to-dextrin ratio from 1:2500 to 1:10000, wherein 1 gram of saidamorphous insulin powder includes from 10 IU to 40 IU of insulin, andfurther wherein when reconstituted in deionized water at a concentrationof 0.03 g of amorphous insulin powder per 1 ml water, a pH of a formedsolution is 6.0-7.6.
 2. The composition-of-matter of claim 1, furthercomprising sodium chloride at a g/g sodium chloride-to-dextrin ratiofrom 1:100 to 1:250 and sodium chloride-to-insulin ratio from 1:20 to1:50.
 3. The composition-of-matter of claim 1, wherein said dextrin ismaltodextrin.
 4. The composition-of-matter of claim 1, wherein saiddextrin is cyclodextrin.
 5. An infant nutrition powder comprising thecomposition-of-matter of claim
 1. 6. The composition-of-matter of claim1, wherein said insulin is sequestered within cavities of said dextrinparticles.
 7. A method of preparing an amorphous insulin powder solublein water comprising: (a) solubilizing insulin in hydrochloric acid toobtain solubilized insulin; (b) diluting said solubilized insulin withwater and adjusting a pH to 6.9-8.0. (c) dissolving dextrin in water andadjusting pH to 7.0-7.5 (d) adding insulin solution (b) to dextrinsolution (c) to thereby sequester said insulin in cavities of saiddextrin molecules; (e) adjusting pH of solution (d) to 6.9-8.0; and (f)freeze drying the solution of (e) to obtain the amorphous insulinpowder.
 8. The method of claim 7, further comprising compounding aresult of (b) or (e) with a sodium chloride solution and a maltodextrinsolution at pH of 6.7-7.5 and adjusting said pH to 6.7-7.6.
 9. Themethod of claim 8, further comprising filtering a result of saidcompounding and lyophilizing a filtrate to obtain an amorphous insulinpowder with a pH 6.0-7.6 when reconstituted in deionized water at aconcentration of 0.03 g of amorphous insulin powder per 1 ml water.